Celiac disease, also known as celiac sprue, nontropical sprue,
gluten-induced enteropathy, or gluten-sensitive enteropathy (GSE), is a
chronic inflammatory disorder of the small intestine characterized by
malabsorption after ingestion of wheat gluten or related proteins in rye
(secalins) and barley (hordeins) in individuals with a certain genetic
background. (1-3) The pathogenesis involves a T-cell-mediated immune
response and autoreactive B lymphocytes that produce autoantibodies
directed against gliadin, endomysium, or tissue transglutaminase in
individuals with a genetic susceptibility related to human leukocyte
antigens HLA-DQ2 and HLA-DQ8. (4-6) The classical textbook description
of celiac disease with malabsorption syndrome and associated flat mucosa
comes from previous studies when the initial peroral jejunal biopsies
revealed the typical advanced mucosal lesion. It is probably useful
within the context of this review that the term celiac disease be
restricted to that definition and that the remaining body of the
iceberg--comprising the latent celiac disease, the monosymptomatic
patients, and relatives of patients with celiac disease showing milder
mucosal pathology--be discussed under the wider term of gluten-sensitive
enteropathy or simply gluten sensitivity.

DIAGNOSIS

With the description of the spectrum of mucosal pathology and the
availability of genetic and highly reliable serologic markers, a
substantial change has occurred in the diagnosis of GSE. Most
importantly, the presence of total flat mucosa, with no detectable villi
on its surface, is no longer necessary provided other histologic
features of GSE and specific antibodies are present in a patient with
genetic susceptibility. (7) Although diagnosis currently relies on
clinicopathologic studies including mucosal biopsy, serologic tests, and
the effects of a diet free of gluten on the symptoms, there is still a
need for consensus guidelines set by the major scientific organizations
associated with GSE. According to the revised criteria of the European
Society for Paediatric Gastroenterology and Nutrition, (8) an initial
characteristic small-intestinal abnormality should be regarded as
essential and there should be a clear-cut response to strict gluten-free
diet (GFD) with clinical recovery within weeks. In asymptomatic
patients, a second follow-up biopsy under a GFD is advised to
demonstrate the histologic recovery of the mucosa, which usually does
not develop before 6 months. (9) The American Gastroenterological
Association mandates a biopsy to confirm the diagnosis in people
suspected of having GSE, (10) whereas the US National Institutes of
Health, in its recent consensus statement, recommends biopsies only
after a positive serologic finding or when serologic results are
nondiagnostic. (11) In summary, the diagnostic scheme of GSE consists of
the following:

Symptomatology of GSE is not associated with the severity of the
mucosal lesion but is mainly related to the length of the affected
bowel. (14-16) Clinical presentation varies from full-blown
malabsorption with weight loss, diarrhea, and steatorrhea to more subtle
symptoms such as folate- or iron-deficiency anemia, flatulence, episodic
diarrhea, loose stools, neurologic problems, osteoporosis, and vitamin K
and D deficiencies in as many as 50% of patients. (2,3,17) It also
varies with the age of the patient, the duration of the disease, and the
presence of extraintestinal findings. (12) In children, usually within
few months of introducing the child to wheat-based foods, the classic
syndrome of chronic diarrhea, steatorrhea, abdominal distension, and
failure to thrive appears between 6 months and 2 years of age. (14,18)
Both weight (40% below 10th centile) and growth (25% below 10th centile)
are affected in these children, although weight is affected more often.
(19) Other children with fewer symptoms may escape detection and will
perhaps not be diagnosed until adulthood. (9) If undiagnosed, however,
GSE can lead to permanent short stature and pubertal delay as well as
the sequelae of nutrient deficiency such as iron deficiency or
megaloblastic anemia and rickets. (9,19,20) Patients presenting with
symptoms as adults may also have short stature and/or historical
symptoms indicative of ongoing disease since childhood. Current
estimates show that the incidence of GSE is 1:200 or higher in
wheat-eating populations such as Western Europe and North America, while
the incidence continues to rise in Eastern societies, possibly as a
result of "western-style" eating habits. (14) Prompt diagnosis
and treatment of GSE not only eases symptoms and improves quality of
life but also has the potential to decrease long-term risks for
lymphoma, gastrointestinal cancers, dermatitis herpetiformis,
osteopathy, endocrine abnormalities, infertility, cardiomyopathy, and
other autoimmune disorders. (14,17,21)

Latent Gluten Sensitivity

The term latent gluten sensitivity is used to descibe symptoms in
patients with evidence of gluten sensitivity but without full-blown
symptoms of sprue. (22-25) Terms that have been applied to such cases
include subclinical, silent, or occult celiac disease; gluten-sensitive
disease with mild enteropathy; low-grade enteropathy; minimally
symptomatic enteropathy; and potential celiac disease. (25-29) This form
of GSE is characterized by the presence of no or only mild changes in
the proximal small-intestinal mucosa, subtle symptoms when investigated
carefully, and isolated positivity of serologic tests. These cases prove
to be difficult to diagnose because of the atypical symptoms that do not
immediately suggest a gastrointestinal cause. Indeed, almost half of
individuals with latent gluten sensitivity may be apparently well, while
others may have a sense of malaise that is difficult to define. Such
cases require vigorous histopathologic evaluation, as the findings may
occur along a spectrum of normal mucosa with increased numbers of
intraepithelial lymphocytes (IELs) to flat mucosa, and they should not
be missed since patients still remain predisposed to cancer or lymphoma
by virtue of chronic gluten ingestion. (22,23,27-29)

Serologic Tests

Widespread availablity of serologic tests has permitted physicians
to test their patients for GSE without further aid from pathology.
Serologic tests are based on the use of immunoglobulin (Ig) A isotypes
and include antigliadin antibodies as well as connective tissue
antibodies such as reticulin antibodies, endomysial antibodies (EMAs),
and tissue transglutaminase (tTG) antibodies. (7,30-33) In most
patients, serologic positivity supports the diagnosis and these tests
are very useful for screening and follow-up. (7,32-34) The enzyme-linked
immunosorbent assay-based clinical test for IgA anti-tTG antibodies has
a high sensitivity and specificity and has recently become the serologic
test of choice for GSE, largely replacing other antibody tests. (30,31)
While EMA is as sensitive and specific as tTG, the immunofluorescent
test used for detection of EMA is time-consuming and more subjectively
interpreted than the tTG test. (32,33) Serologic positivity usually
correlates with the degree of mucosal damage, while minimal histologic
lesion of intraepithelial lymphocytosis (IELosis) often presents with
normal serologic findings. (7,35-37) In parallel with this view, a
previous study (34) demonstrated that increasing titers of tTG predicted
higher levels of villous flattening. Similar findings were observed in
pediatric patients who had tTG levels above 100 units and showed
advanced (Marsh type 3) lesions in their biopsy specimens. (34,38) Taken
together, these findings indicate that a negative serologic result is
not sufficient to rule out GSE with a Marsh type 1 lesion.

Human leukocyte antigen (HLA) testing to detect susceptible HLA
subtypes is also used in the routine diagnostic workup of GSE. (7,39)
While HLA-DQ2 is found in 90% to 95% of patients with GSE, most of the
remaining cases are associated with HLA-DQ8. Since these HLA alleles are
found in up to 40% of the general population, their presence does not
aid the diagnosis directly, but the absence of DQ2 or DQ8 virtually
excludes GSE. (7) It is therefore justified to state that, despite the
development of new serologic tests and genetic analysis,
small-intestinal biopsy continues to be the gold standard for the
diagnosis of GSE, particularly in less severely affected patients with
mild mucosal abnormality.

Small-Intestinal Biopsy: Site and Number?

Similar to its wide variation in clinical manifestations, GSE has a
wide spectrum of histologic abnormalities, which makes interpretation of
small-intestinal biopsy specimens problematic for the pathologist. The
damage to the small-intestinal mucosa classically involves the proximal
small intestine including duodenum and upper jejunum and extends
distally for a variable length into the ileum. (14,40,41) Healing of the
small-bowel mucosa, on the other hand, takes place in a distal to
proximal direction. (15) This may take at least 6 months and may even be
prolonged up to 24 months after treatment with GFD. (42) Because of the
slow tempo of IEL ([gamma][delta] subtype in particular) loss from the
epithelium and entry after gluten ingestion, IEL count may be the last
feature to return to normal after a GFD. (41,43)

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The number of small-intestinal biopsies has substantially increased
over the years, partly because of the increased awareness by clinicians
of the atypical forms of GSE, as well as the increased use of upper
gastrointestinal endoscopy. Previously, small-intestinal biopsies were
always taken by suction capsule, conventionally positioned at the distal
duodenum, duodeno-jejunal junction (ligament of Treitz), or proximal
jejunum under fluoroscopic control. (12,14,16) Although, capsule
biopsies are usually bigger and easier to orientate, swallowing the
capsule is discomforting for the patient and is more labor-intensive and
time-consuming for the physician. In the past decade, duodenal biopsies
have almost entirely replaced capsule biopsies of jejunal mucosa for the
diagnosis of GSE in most gastroenterology units, essentially because
endoscopy has the advantages of saving time and reducing the risk of
failure and false-negative findings. (12,44-48) Also, multiple targeted
biopsies can be taken during endoscopy because, in most patients with
flat mucosa, the duodenum shows typical endoscopic features described as
mosaic appearance, scalloping, or reduction of duodenal folds.
(12,14,45,46) Newer endoscopic methods, such as push enteroscopy and
double-balloon enteroscopy, allow access to the entire length of small
bowel, (15) but are more timely and costly compared with upper
gastrointestinal endoscopy. Obtaining biopsy samples of adequate size
from across a circular mucosal fold is as important as the biopsy site
itself. (12,14,15) Pathologists should be aware that biopsy forceps can
crush and destroy tissue, causing hemorrhage in the sample and thus
making the evaluation of specimen difficult. Superficial biopsy samples
lacking muscularis mucosa can cause separation of the villous bases,
resulting in shorter and thicker villi that can easily be misinterpreted
in favor of a diagnosis of GSE.

Conflicting reports exist in the literature regarding the
distribution pattern of mucosal pathologic features along the small
bowel. (16,49-51) Although it was widely accepted in the past that
villous flattening rarely coexisted with histologically normal mucosa,
currently, many investigators believe that GSE can exhibit a patchy
distribution, that is, areas with villous flattening may occur in
proximity to areas with mild villous shortening and also in areas with
normal histologic features, particularly in pediatric patients. (50,51)
This notion may lead to false-negative diagnosis, particularly when
there is inadequate sampling. However, the optimal number of biopsy
specimens necessary to confirm the diagnosis of GSE is still not known.
There are no recommendations in the guidelines of the North American
Society for Pediatric Gastroenterology, Hepatology and Nutrition, (52)
while the American Gastroenterology Association has recommended 6 biopsy
specimens as necessary for the diagnosis of GSE. (53) In practice, it
seems reasonable to suggest that at least 4 endoscopic biopsy samples
must be taken from distal duodenum with 2 samples from the bulbus to
detect patchy and subtle mucosal lesions in GSE. Biopsy specimens from
the bulbus, however, should be interpreted with caution because this
area is susceptible to peptic injury and contains prominent Brunner
glands, which can lead to obliteration of the villi, with an
artifactually flat mucosal appearance. (12,47,54)

What Is "Normal"?

Pathologists should be capable of recognizing normal features of
small-intestinal mucosa so as to interpret the abnormalities associated
with GSE correctly. It is generally accepted that the presence of at
least 3 or 4 consecutive villi with a normal villous to crypt ratio in a
biopsy sample is sufficient to consider as normal. (12,15) Normal
small-intestinal mucosa has long, slender villi, with this ratio ranging
between 3:1 to 5:1 depending on the site of the biopsy; shorter villi
are found in the duodenum, whereas the height of the villi increases
distally from jejunum to ileum (Figure 1). Normal distribution of IELs
along the villi shows a characteristic decrease from the base of the
villus toward the villous tip and resembles the musical
"decrescendo" sign. (55) However, the pathologist should be
aware of the possible patchiness of mucosal lesions when making a
decision of normal mucosa and ruling out GSE.

For accurate histopathologic assessment of villus to crypt ratio,
it is essential that the biopsy specimen be properly oriented with the
luminal surface upwards such that the villi should be vertical to the
muscularis mucosae (Figure 2). (12,13,15) Specimens can be oriented on a
supporting medium (eg, a strip of filter paper, a piece of tissue, or
dental wax) with the naked eye or with the assistance of a dissecting
microscope; embedded in wax; and cut through vertical plan.13 In
tangentially cut sections, artifactual shortening of the villi and
falsely increased lymphocytes in the surface epithelium can occur.
(12,14) The approach to biopsy specimen by the pathologist is as
important as the correct orientation and involves low-power microscopic
examination for architectural abnormalities, as well as cellular
distribution, and is followed by a high-power view to assess cellular
content and epithelia. Although the exact numbers are not known,
fewinflammatory cells comprising plasma cells, lymphocytes, eosinophils,
and macrophages are found in the lower one-third of the lamina propria,
while villous lamina propria is considered as "empty" in
normal small-intestinal mucosa. (12,15) In the presence of inflammation,
however, inflammatory cells, including polymorphonuclear leukocytes,
infiltrate upper parts of the lamina propria and cause obliteration of
the villi.

THE MORPHOLOGIC SPECTRUM OF GSE

Histologic evidence of GSE depends on abnormalities in either
architecture (villous shortening and crypt hyperplasia) or the number of
IELs or both. In its classical form, GSE results in shortened, widened
villi or even totally flat mucosa with hyperplastic crypts. Overall
thickness of the mucosa remains relatively unchanged, but villous to
cyrpt ratio (normally 3:1 in distal duodenum and 2:1 in bulbus)
decreases as the villi become shortened. These architectural changes are
preceded by an increase in the number of IELs as compared to normal
numbers, corresponding to the cell-mediated immune nature of the
disorder. It is this group of cases that should be actively sought in
biopsy specimens in which GSE is part of the clinical differential
diagnosis. (14,56)

Marsh (56) was the first to describe the morphologic continuum of
GSE, which ranges from normal villous architecture with IEL increase as
the only abnormality to flat mucosa with crypt hyperplasia, increase in
the number of IELs, epithelial destruction, and increased lamina propria
inflammation. Marsh classification will be discussed in detail in
association with other classification schemes in the following sections.

Intraepithelial Lymphocytes

Intraepithelial lymphocytes have been considered to be responsible
for the epithelial damage observed in GSE, although the exact mechanism
is still not known. Most IELs are T lymphocytes, which are mostly
cytotoxic T cells expressing [alpha][beta] T-cell receptor (TCR) on
their surface. The population specifically expanded in GSE is the
[CD3.sup.+]/ [CD4.sup.-]/[CD8.sup.-], [gamma][delta] TCR-bearing IELs,
which is only 5% of the total in normal mucosa. (57,58)

Over the years, the cutoff value, and thus the normal number of
IELs, has been subject to major variation and a significant reduction
has occurred in the highest value for normal. The trend toward a lower
normal number of IELs is reflected by the decreasing numbers of IELs,
including 25, 22, or even 20 IELs per 100 epithelial cells, incorporated
into revised classification schemes. (59-62) The reasons for this can
partly be attributed to the change in biopsy site (see previous
sections)--normal jejunal mucosa has a higher number of IELs than
duodenal mucosa--and also to the decrease in section thickness over the
years and to the application of immunohistochemistry to define IELs.
Currently, the normal upper limit of IELs is accepted as 20 lymphocytes
per 100 enterocytes (a ratio of 1 IEL per 5 enterocytes) in
hematoxylin-eosin sections, whereas 25 IELs per 100 enterocytes (or a
ratio of 1:4) is considered the upper limit of normal in
CD3-immunostained slides. (62) It remains, however, to be determined
whether lowering the upper limit for IELs will adversely affect the
specificity of small-bowel biopsy in the diagnosis of GSE, since it may
cause overlaps with other causes of IELosis. At this point, it would be
wise to state that the greater the number of duodenal biopsy samples
seen by the pathologist, the more accurately will be defined the normal
range of IELs.

To Count or Not To Count?

Although there are various ways of enumerating IELs, almost all are
impractical for incorporation into the routine pathology practice.
Counting IELs per 100 enterocytes (usually in a total of 300-500
epithelial cells) has been the most widely used method, either with the
aid of CD3 immunostaining or without. (56,57,60-65) Recently, an
alternative method of screening for GSE has been proposed in which IEL
counts in villous tips (5 well-oriented villi, 20 enterocytes at the tip
of each) can be used for a rapid assesment. The normal average number of
IELs according to "villous-tip" method is less than 5 per 20
enterocytes, while counts between 6 to 12 per 20 epithelial cells are
considered suggestive of GSE. (66,67) Routine application of CD3
immunohistochemistry has been suggested as a better means of evaluating
the number and distribution of IELs when there is normal villous
architecture in a biopsy specimen. (62,68) In my experience,
immunohistochemistry may, indeed, be very useful when there is a
suspected, rather than a definite increase in IELs. In practice,
however, I strongly believe that the distibution pattern of IELs within
the epithelium is more valuable than the actual counts, since accurate
quantification can be difficult because of nuclear overlap and
resemblance of IELs to enterocyte nuclei and granulocytes. Patients with
GSE, including those with normal villi in their biopsy specimen, lack
the normal decrescendo pattern as a result of increased density of IELs
in the distal tips of the villi, thus causing a diffuse infiltration of
the villous epithelium. (55,67-69) Therefore, a practical approach would
be to scan the villi and look for the loss of normal decrescendo pattern
or the presence of a diffuse and uniform infiltration. This approach
would also serve to correctly interpret cases with mild increases in IEL
numbers that may overlap with those of healthy individuals. It should be
stressed, however, that the loss of normal distribution pattern is not a
diagnostic, but a suggestive feature of GSE. In my experience, as well
as that of others, (66,68) immunohistochemical staining with CD3 helps
to highlight the distribution pattern of IEL within the villous
epithelium, though there are some contradictory reports (55) in the
literature (Figure 3, A and B).

Causes of IELosis

Intraepithelal lymphocytes are active components of the mucosal
immune system, which undergoes threat from luminal antigens such as
gluten, microorganisms, drugs, and other toxic molecules, all of which
can cause an increase in IEL numbers. Although IELosis in a normal
mucosa is an increasingly reported pathologic feature in GSE, it is by
no means diagnostic, since overlap in the IEL counts occurs between
patients with and without GSE. Various pathologic processes, including
food allergies other than GSE, Helicobacter pylori-assocated duodenitis,
giardiasis, graft-versus-host disease, tropical sprue, viral enteritis,
injury caused by nonsteroidal anti-inflammatory drugs,
chemoradiotherapy-induced enteritis, autoimmune enteropathy,
immunodeficiencies, and Crohn disease can induce IELosis with or without
associated architectural changes. (12,14,15) Some of these entities are
discussed in the differential diagnosis section and are listed in Table
1.

[FIGURE 3 OMITTED]

Lamina Propria

Although considerable amount of research has focused mainly on
IELs, lamina propria seems to have an even more important part in the
pathogenesis of GSE. The presentation of gliadin peptides by
antigen-presenting cells bearing DQ2 or DQ8 to lamina propria helper T
cells leads to T cell activation and secretion of various cytokines.
These events take place in the lamina propria and are considered to
precede IEL infiltration. (56) Because of the immunopathologic basis of
the disease, the pathologists encounter a complex and heterogenous
population of inflammatory cells in the lamina propria comprising plasma
cells that locally produce antigliadin and EMAs in addition to T cells
that include predominantly helper T cells as well as a few cytotoxic
cells. Also, neutrophils, eosinophils, and mast cells may be found in
varying numbers, although grading this inflammatory reaction is
difficult and impractical. (15) Since none of these changes is specific
to GSE, IELosis and architectural changes affecting villous to crypt
ratio remain as the main diagnostic parameters of pathologic evaluation.

CLASSIFICATION OF GSE

On the basis of substantial amount of clinical research, including
a sequence of dynamic studies, Marsh (56) first introduced the
morphologic continuum of gluten sensitivity. Marsh classification is
composed of 4 consecutive states of mucosal damage (types 1-4) as
follows.

Infiltrative lesion: Increased numbers of IELs in the villus
epithelium in an otherwise normal mucosa with normal villous to crypt
ratio (Marsh type 1). A type 1 lesion may be observed in patients with
GSE who follow a GFD and indicate that minimal amounts of gliadin are
still ingested or that the patient is not yet in full remission. More
importantly, it is found in latent GSE, in family members of patients
with GSE (potential candidates for celiac disease), and in some
individuals with dermatitis herpetiformis. (22,35,55,56,61) An increase
in the number of IELs is the first and most sensitive index of gluten
effect on the mucosa and, although not specific, is the most
characteristic histologic feature of GSE. (70) Gluten sensitivity has
also been known to affect other parts of the gastrointestinal tract,
including esophagus, stomach, and large intestine and, interestingly
enough, IELs are also increased in these areas. (71,72) In accordance
with this information, rectal biopsy, performed before and after a
rectal gluten challenge, (73-75) was proposed as a simpler means of
diagnosing GSE. However, it has not gained international acceptance as a
useful clinical tool and thus, pathologists are currently unlikely to
receive rectal biopsy specimens taken for the diagnosis of GSE other
than for research purposes.

Hyperplastic lesion: Crypt hyperplasia with normal villi showing
increased numbers of IELs (Marsh type 2). The importance of this lesion
is that it was first described by Mowat and Ferguson (76) in a neonatal
mouse model of graft-versus-host disease. It therefore clearly
represents another distinctive, T-cell-mediated immune response of
intestinal mucosa. Type 2 lesion is only very rarely, if ever,
encountered in the biopsy samples of patients with GSE and has mainly
been observed under experimental conditions or time-dose-related gluten
challenge studies. (77-81) The results of these studies demonstrated
that crypt hyperplasia was the first architectural change in the
evolution of the mucosal lesion, initiated by the increase in IELs.
Crypt hyperplasia or elongation of the length of the crypt is a process
that precedes villous shortening despite the general misconception that
it is a compensatory feature occurring in response to villous
shortening. (77) Elongation may be caused by the interaction of stromal
cells with the epithelium through the secretion of various cytokines and
influx of inflammatory cells releasing growth factors. In the advanced
stages of the disease, matrix metalloproteinases and their tissue
inhibitors may also play a role in the development of mucosal
architectural changes through their degradative action on the stromal
cells of the lamina propria. (12,15,56,70)

Although I believe that the original type 2 (hyperplastic) lesion
clearly exists within the spectrum of gluten sensitivity, in routine
practice we do not see these lesions in the formthat theywere originally
described byMarsh. In his schematic drawings, the type 2 lesion has
always been illustrated as normal villi with elongated crypts.
(21,78,82) In practice, however, when there is crypt hyperplasia, it is
always accompanied by shortened villi. Similarly, for treated patients
with celiac disease, one observes restoration of villi (still short,
though) together with crypt hyperplasia, which again is different from
Marsh's original type 2 lesion. (41,77) This led to a confusion
among pathologists who have (naturally) interpreted Marsh type 2 lesion
as mucosa containing shortened villi and hyperplastic crypts, simply
because this pattern is what they see in biopsies. Indeed, in many
articles published by other authors, (7,14,49) the photographs
illustrating Marsh type 2 lesions are those of mucosae with shortened
villi and crypt hyperplasia, in contrast to Marsh's original
definition. (56) This type of lesion occurs only in strictly
time-dose-dependent gluten challenge studies, where it is possible to
witness mucosal abnormalities in a more dynamic manner. In one such
study, (77) in which jejunal biopsy was performed before and at 12, 36,
60, and 84 hours after challenge during a 5-day time course, the entire
spectrum of mucosal changes was reproduced initiating with the
infiltrative lesion in which dose-dependent lymphoid infiltration into
normal villous epithelium had occurred. This was followed by enlargement
of crypts to produce the infiltrative-hyperplastic lesion without any
villous shortening. Larger challenges produced villous shortening
followed by flattening with further increases in crypt size. (77)

Atrophic lesion: Flat mucosa with crypt hypoplasia and mild
inflammation (Marsh type 4). This is the very rare hypoplastic lesion
that is characterized by a flat mucosa with only a few crypts and normal
IEL counts. It is usually observed in patients with refractory sprue or
patients who develop enteropathy-induced T-cell lymphoma (EITCL).
(56,70)

Despite the fact that Marsh is a gastroenterologist, his efforts to
understand the mucosal pathology of GSE have made the most powerful
impact on the interpretation of small-intestinal biopsies by
pathologists. His classification has not only enhanced our understanding
of the biology of the disease but has also facilitated the recognition
of cases with increased IEL counts in an otherwise normal mucosa. One
problem arising from Marsh classification is that the cutoff for the
number of IELs has not been defined. This is mainly because of the
strict morphometric techniques that Marsh used to enumerate IELs in his
studies, as well as other cells of interest in small-intestinal mucosa.
Cells were enumerated with respect to a standard test area of [10.sup.4]
[micro][m.sup.2] of muscularis mucosae in order to define the absolute
numbers in a 3-dimensional fashion, (65,77,79-81,83) which, in
day-to-day practice, is far too time-consuming and unpractical to be
applied to routine pathology. The updated classification, (59) proposed
after 4 years, has largely overcome this problem by indicating that 40
IELs per 100 enterocytes should be the cutoff point for normal, a figure
possibly derived from jejunal mucosal biopsies, which used to be taken
previously for the diagnosis of GSE. Section thickness may have also
contributed to the high IEL counts in the past, since sections 3- to
4-[micro] thick are taken in most pathology laboratories now. Upper
limit of IELs, on the other hand, is 25 per 100 enterocytes in the
duodenal mucosa, which is currently the preferred biopsy site for the
diagnosis of GSE. Emphasis made on milder mucosal lesions by defining
upper limits of normal IEL counts is a valuable impact of the Oberhuber
classification, although, in routine practice, it is not always
necessary nor possible to enumerate the IELs; rather, an estimate is
made by looking at their distribution within the epithelium.

The second revision in this classification was the subgrouping of
type 3 lesion (flat mucosa) into 3 grades with regard to the severity of
villous shortening ("atrophy" in the original report). The
authors (59) have classified Marsh type 3 lesions as follows:

Type 3B: Marked villous atrophy. Marked villous flattening
indicates that only short tentlike remainders of the villi are present.

Type 3C: Completely flat mucosa. Total villous flattening or flat
mucosa indicates that no more villi can be recognized and that the
surface is flat.

Although the Marsh classification, as modified by Oberhuber et al,
(59) is used by many pathologists, both for diagnosis and also to assess
the response to therapy, this classification has the potential to cause
significant reproducibility problems leading to increased intraobserver
and interobserver variations because of the greater number of diagnostic
categories. Moreover, definitions of type 3A lesions (as mild or
moderate villous shortening) and type 3B lesions (as marked villous
shortening) lack objective criteria with respect to villous to crypt
ratio, thereby increasing the subjectivity of interpretation.

Recently, a new and relatively simpler classification was proposed
by Corazza and Villanaci. (84) Their 3-grade classification maintains
the original type 1 (infiltrative) lesion as grade A, while type 2
(hyperplastic) lesion is totally left out by the authors, stating that
it has no additional use for diagnosing cases that would already be
identified by the increased IEL counts. Oberhuber type 3A and 3B lesions
are grouped into a single grade as grade B1, whereas type 3C, the flat
mucosa, is maintained as grade B2. Marsh type 4 (atrophic) lesion has
been made obsolete by the recent finding that refractory sprue,
ulcerative jejunitis, and EITCL are all characterized by aberrant clonal
T-cell expansions, as demonstrated by immunohistochemical and molecular
techniques. (12,85-88) In summary, Corazza and Villanaci (84) have
proposed that lesions characterizing GSE should be classified as
nonatrophic (grade A) and atrophic (grade B) and that grade B lesions
should be split into (1) grade B1, characterized by a villous to crypt
ratio of less than 3 to 1, and (2) grade B2, characterized by completely
flat mucosa with no detectable villi. Although there are no obvious
problems with this proposal, I would object to the use of the term grade
to classify the mucosal lesions, since the severity of mucosal lesion
does not necessarily indicate clinical severity. Moreover, the term
grade is used for tumor differentiation, thereby, making it
inappropriate for classifying the mucosal pathology of GSE. I would
personally suggest that we retain the term type, not only because it is
accepted both by the gastroenterologists and pathologists, but also as a
tribute to Marsh. I would also object to the use of the term atrophy to
define villous architectural changes, as dynamic studies in the past
have shown that the mucosa does not at all undergo a process of atrophy,
but rather it demonstrates a hyperplastic state characterized by
elongation of the crypts and widening of the lamina propria by
inflammation. This observation is also supported by the finding that the
height of the mucosa is not changed when an infiltrative lesion
progresses to a flat lesion. (77-81) Highlighting the term atrophy as
the major feature of immunologically driven pathologic processes that
are induced by gluten fails to include the condition of most patients,
who often present with milder lesions devoid of "atrophy," or
more appropriately, villous flattening. (56,70)

The "New" Version of an "Old" Classification

The above discussion highlights the problems regarding the
classification systems currently used for GSE. The growing number of
duodenal biopsies taken for suspected GSE, however, potentiates the
pathologist's need for a more reproducible and standardized
classification, which is equally appealing to clinicans. At this point,
I would like to propose an updated version of the original Marsh
classification, which, from a pathologist's point of view, is much
simpler and user-friendly. In the proposed classification, mucosal
pathologic features will be defined in 3 groups, mainly depending on the
degree of cellular and architectural abnormalities.

Type 1: Normal villi with IELosis (Figure 4, A). This type
corresponds to Marsh type 1, also present in the Oberhuber
classification, and to grade A in Corazza and Villanaci's proposal.

Type 2: Shortened villi (<3:1 or <2:1 in bulbus) with IELosis
and crypt hyperplasia (Figure 4, B). This type corresponds to types 3A
and 3B in the Oberhuber classification and to grade B1 in Corazza and
Villanaci's proposal.

Type 3: Completely flat mucosa with IELosis and crypt hyperplasia
(Figure 4, C). This type corresponds to Marsh type 3, and also to type
3C in the Oberhuber classification, and to grade B2 in Corazza and
Villanaci's proposal.

While types 1 and 3 are exactly identical to their counterparts in
the original Marsh classification, type 2, in the current proposal,
comprises cases with crypt hyperplasia and any degree of villous
shortening but not flattening (ie, villi can still be identified). As
already stressed, villous shortening must be evaluated according to the
biopsy site, since the normal villous to crypt ratio varies throughout
the small intestine. Thus, types 2 and 4 of the original Marsh
classification have been made obsolete in this proposal because of the
reasons already discussed in the previous paragraphs. The similarity of
this new version to the original Marsh classification may help to gain a
wider acceptance from both pathologists and clinicians; however, further
validation through clinicopathologic studies is necessary. Comparison of
all 4 classifications is presented in Table 2.

VARIANTS OF GSE

Patients with incomplete or no response to GFD are considered
within the context of refractory sprue. Diagnosis of refractory sprue is
rarely made and should be strictly limited to patients with clinical
features of GSE not responding to GFD for at least 6 months. It can be
either primary, as lack of initial response to diet, or secondary, as
unresponsiveness to diet in the form of a relapse. Primary refractory
sprue can include many different pathologic conditions mimicking GSE,
comprising collagenous sprue, ulcerative jejunitis, and EITCL. (85,86)
Histopathologically, most cases with refractory sprue show flat or near
flat mucosa with a dense mononuclear infiltrate of mainly plasma cells
and lymphocytes in the lamina propria and a massive increase in IELs
(Figure 5, A). The lymphocytes in the villous, crypt epithelium, and
lamina propria are normal to medium in size, with a normal cytologic
appearance (Figure 5, B). However, most cases of refractory sprue have
abnormal T-cell phenotypes ([CD103.sup.+] but
[CD4.sup.-]/[CD8.sup.-]/[TCR.sup.-]) showing clonal expansions as proved
by monoclonal reaarangements of the TCR [gamma][delta] gene. These
features have led to the idea that such cases represent a form of in
"situ" T-cell lymphoma. (12,15,85-90) Collagenous sprue refers
to a variant entity having a thick collagen table beneath the surface
epithelium of a mucosa, which also has other typical features of GSE,
including IELosis. (12,14,15,91)

Patients with long-standing malabsorption can develop EITCL as the
most fearsome complication of GSE. They usually suffer from severe
malabsorption refractory to GFD, causing weight loss and complications
such as perforations and bleeding due to ulcerative lesions.
Histopathologically, the mucosa is flat with ulcerations. An atypical
population of neoplastic lymphocytes infiltrate the mucosa. Lymphoma
cells are pleomorphic large cells that are double-negative ([CD8-.sup.-]
and [CD4.sup.-]) for cell surface markers, but almost always positive
for CD30, which is also associated with poor prognosis. (85-90)

DIFFERENTIAL DIAGNOSIS

Differential diagnosis of GSE involves a great variety of disorders
with architectural abnormalities and/or IELosis (see Table 1). Among
these, only conditions that are likely to be encountered by the
pathologist in routine duodenal biopsies will be briefly mentioned here.

Chronic H pylori-associated duodenitis: Helicobacter pylori
gastritis can cause an increase in IELs in duodenal bulb, which causes a
major diagnostic confusion with GSE. This is particularly a problem when
distal duodenum is not biopsied, since it is usually normal in H pylori
infection. (54) Helicobacter pylori colonizes the duodenal mucosa only
in areas of gastric foveolar metaplasia, which can lead to active
duodenitis. (92-94) Features useful in distinguishing this condition
from GSE are the heavy neutrophilic infiltration of the lamina propria,
as well as the surface epithelium; relatively less architectural damage
in terms of villous shortening; and the presence of foveolar metaplasia,
though the latter may also be seen in GSE if the biopsy is taken from
the bulbus affected by H pylori (personal observation).

[FIGURE 4 OMITTED]

Food allergy: Hypersensitivity to food antigens other than gluten,
including cow's milk, soy protein, fish, rice, and chicken may also
be associated with increased numbers of IELs, as well as architectural
changes in the form of patchy or diffuse disease in any part of the
gastrointestinal tract in affected individuals. Lymphoid hyperplasia in
the bulbus is a common feature of food allergy together with
eosinophilic infiltration of the lamina propria and IELosis, especially
in children, while villous shortening is only rarely encountered.
(14,95,96)

[FIGURE 5 OMITTED]

Infections: A wide variety of infectious agents including viruses,
parasites, and bacteria can affect small-intestinal mucosa. Since it is
beyond the scope of this review to discuss these in detail, a general
view will be given. Most infections cause only mild and nonspecific
alterations in the intestinal mucosa including IELosis, lamina propria
inflammation, and rarely, architectural changes, whereas some, such as
giardiasis; Whipple disease; opportunistic infections including
microsporidiosis, cryptosporidiosis, and CMV can be diagnosed by
identifying the microorganism in biopsy specimens. Mucosal histologic
features in bacterial overgrowth, either due to gastric hypochlorhydria
or dysmotility, may be normal but often show architectural changes as
well as increased IEL counts in a patchy fashion. (12,14,97,98)

Autoimmune enteropathy: This condition, caused by autoantibodies to
enterocytes, primarily affects children. Histologic findings of villous
flattening and crypt hyperplasia are similar to GSE, but the resultant
secretory diarrhea is unresponsive to GFD or total parenteral nutrition.
There may be some degree of IEL increase, but neutrophils are more
prominent in the surface epithelium. (12,14,15,99)

Crohn disease: Although the exact frequency of upper
gastrointestinal involvement in Crohn disease is not known, it can
represent up to 30% to 50% of cases in some centers. Granulomas are
uncommon, but when found, they are usually more often in the stomach
than the duodenum and can be helpful in the differential diagnosis.
(12,14,100,101)

REPORTING

Histopathologically, the manifestations of GSE display a range in
severity. Also, a long list of entities cause a pathologic picture
similar to that of GSE, thereby complicating the histopathologic
diagnosis and the pathologic reporting. I believe that the
small-intestinal biopsy is essential in the diagnosis of GSE and that a
pathology report should always be descriptive unless the serologic and
clinical findings are indicative of GSE. Considering that there are many
entities in the differential diagnosis, the pathologist should always
communicate with the clinician and obtain serologic and clinical data
before making a definite diagnosis. (12,14,15) It is important that
pathologists recognize the biopsy site, which may alter their
interpretation of what is "normal," and that they report the
most severely affected part of the biopsy specimen, as the lesion may
show some patchiness. Therefore, the pathology report should include the
biopsy site and number of biopsy specimens, a description of the
architecture in terms of villous to crypt ratio, the presence of damage
to surface epithelium, evidence of IELosis, and the type and degree of
lamina propria inflammation. Description of mucosal pathologic features
should be followed by a descriptive diagnosis and classification of the
lesion. At the end, a comment on possible diagnosis or differential
diagnosis may also be included. When indicated, the pathologist may
recommend serologic testing, follow-up biopsy, or a second opinion from
an experienced gastrointestinal pathologist.

CONCLUSIONS

Widely varying clinical presentations and mucosal pathologic
features of GSE make the diagnosis difficult for the pathologist.
Although considered as characteristic, IELosis can be observed in a
number of other diseases affecting the small intestine, thereby
complicating the diagnostic picture as there is no cutoff for IEL counts
for distinguishing GSE from other causes of IELosis. Therefore, in
routine workup, the pathologist should not feel obliged to enumerate
IELs, but rather should scan through the biopsy pieces to see whether
there is a definite increase in IELs and whether it is diffuse or absent
from villous tips. CD3 immunostaining is recommended to highlight the
distribution pattern of IELs. Counting is useful when there is no
serologic correlation. It may also help when there is no obvious
increase in IEL counts or when the biopsy specimen is not properly
oriented. An IEL count of more than 20 per 100 enterocytes in
hematoxylin-eosin sections (.25 in CD3 immunostaining) should be
considered as a definite increase and reported as "compatible with
a diagnosis of GSE" if it is diffusely distributed over the villi.

As our understanding of the disease has changed over the years, a
need for modification of the original classification of mucosal
pathology has emerged. A simpler and clarified version, comprising
identical counterparts of original Marsh types 1 and 3 lesions, and the
"new" type 2 lesion, which is redefined as crypt hyperplasia
with shortened but not flattened villi, is proposed in this review. This
proposal is based not only on the vast amount of information available
in the literature but also on the research data from Marsh's
laboratory and from almost 20 years of personal experience in
small-intestinal pathology.

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